In this R00 proposal, Dr. Luca will investigate how molecular recognition influences ligand selectivity in the Notch pathway, a signaling system of fundamental importance to cell fate determination and the pathogenesis of cancer. Dr. Luca will determine the structural and biophysical determinants that distinguish each class of Notch receptor subtype, and will enhance ligand function using techniques rooted in structural and combinatorial biology. Engineered ligands with improved biomedical properties will be evaluated as highly selective Notch antagonists of tumor cell proliferation. Notch signaling is initiated when a Notch receptor on the surface of a cell engages a ligand on an opposing cell, triggering a series of proteolytic cleavages required for activation. While the overall signaling mechanism is conserved, individual Notch receptors and the two classes of Notch ligands, Jagged (Jag) and Delta-like (DLL), are able to induce distinct cellular responses in both normal and cancer cells. There is a large body of literature describing pleiotropic Notch signaling in development and disease, yet the events that govern ligand and receptor-specific effects are poorly understood. Notch-ligand selectivity is further complicated by the ability of Notch receptor glycosylation to inhibit the activity of some ligands and potentiate the activity of others. Structural studies of Notch receptor-ligand interactions, which would provide molecular clarity for this system, are difficult because the nearly undetectable affinity of Notch receptors and ligands precludes reconstitution of stable complexes. Dr. Luca recently overcame this obstacle using directed evolution to affinity-mature Notch1 interactions with Delta-like 4 (DLL4) and Jagged1 (Jag1) to stabilize complexes for co-crystallization. The resulting structures have provided the first visualizations of Notch-ligand interfaces. Here, Dr. Luca will build upon these transformative results by investigating how structural and biophysical parameters influence differential ligand activities. This goal will be achieved by (i) determining the molecular basis by which Notch1 glycosylation inhibits Jag1 signaling, (ii) determining high-resolution crystal structures of Jag1 in complex with Notch2, Notch3 and Notch4, (iii) engineering receptor- and tissue-specific Notch antagonists and (iv) evaluating the therapeutic potential of targeted Notch antagonists to inhibit nonsmall cell lung cancer (NSCLC) tumor growth. As a proof-of-concept, Notch3-specific DLL4 variants and CD24- targeted Notch antagonists will be tested for their ability to inhibit proliferation of NotchhiITGB4+CD24+ tumor propagating cells in a NSCLC model; however, the generation of additional classes of targeted ligands will allow for deep interrogation of Notch biology for years to come. The successful completion of this work will establish the structural and biophysical basis for Notch activation by different receptor and ligand subtypes, and will instruct the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.